On a worldwide basis, malaria is a leading cause of morbidity and mortality; the infection is increasing in incidence and prevalence; and drug-resistant parasites have become commonplace. The need for safe new antimalarials is widely acknowledged to be urgent. Antifolate thymidylate synthase inhibitors are a new class of therapeutic agents, and they have potent antimalarial activity in vitro. Selective toxicity is enhanced by coadministration with thymidine. This project will evaluate the activity of antifolate thymidylate synthase inhibitors in mammalian systems. Test compounds will include those already marketed or in clinical trials, as well as novel compounds. Aim 1 of this project is to evaluate the cytotoxicity of thymidylate synthase inhibitors, with and without concomitant thymidine, against mouse or human cell lines in vitro. Results of these studies, in conjunction with antimalarial potency, will provide a basis for selecting compounds for testing in animals. Treated cells will also be analyzed for intracellular nucleotides, as an indication of the extent of thymidylate synthase blockade and the effectiveness of thymidine protection. In Aim 2, selected thymidylate synthase inhibitors, alone or in combination with thymidine, will be tested against Plasmodium berghei and P. yoelii in mice. Those with greatest potency and selective toxicity will be candidates for further preclinical testing. Aim 3 is devoted to studies of thymidine in humans. Thymidine is a nonessential nutrient in the diet, circulates in submicromolar levels in the blood, and has been used parenterally for over 60 years to treat assorted clinical disorders. Remarkably, despite all this there has been no formal study of orally administered thymidine in humans. As a necessary preliminary to the use of combination therapy in patients with malaria, the bioavailability, plasma levels, and nucleotide concentrations in circulating lymphocytes, will be evaluated in healthy volunteers treated with orally administered thymidine.